Read the high-activity staining and observation of mitochondria and vacuole system today

Experimental principle

In vivo staining is a method of staining that can stain or not poison cells or tissues of living organisms. Its purpose is to show certain structures within living cells without affecting the life activities of the cells and producing any physical and chemical changes that cause cell death. The live dyeing technique can be used to study the cell morphology, physiological and pathological state in the living state.

According to the nature of the dye used and the dyeing method, the living dyeing can be divided into two types: in vivo live dyeing and in vitro live dyeing. In vivo living dyeing is injected into the animal and plant in a colloidal dye solution, and the gel particles of the dye are fixed in some special structures in the cell for easy identification. In vitro live dyeing, also known as super-live staining, is the separation of some cells or tissue fragments from living animals and plants, dyed with dye solution, dyes are selected to be fixed in living cells due to their "electrochemical" characteristics and the dyed parts. It develops in a certain structure. But not any dye can be used as a living body dye. Generally, those non-toxic or less toxic basic dyes (soluble in lipidoids) should be selected and used as a dilute solution. . Jenus Green B and Neutral Red are two important dyes in living dyeing, which have specificity for the staining of mitochondria and vacuole. Jenus Green B can specifically live on mitochondria. Dyeing, this is due to the action of the cytochrome oxidase system in the mitochondria, which keeps the dye in an oxidized state (ie, colored), which is blue-green; in the cytoplasm surrounding the mitochondria, these dyes are reduced to a colorless chromophore. Neutral red is specific for the staining of vacuoles, only the vacuole in living cells is stained red, the nucleus and cytoplasm are not colored, which may be related to certain proteins in the vacuole.

Laboratory supplies

1 Equipment: Microscope, thermostatic water bath, scissors, tweezers, scalpel, slides, coverslips, straws, toothpicks, absorbent paper

2 Reagents:

1Ringer solution: sodium chloride 0.85g, potassium chloride 0.25g, calcium chloride 0.03g

21/5000 Jenus Green B solution: Weigh 0.5g Jenus Green B dissolved in 5ml Ringer solution, slightly heated (30-40 ° C) to dissolve, filtered with filter paper, it is 1% stock solution. 1 ml of 1% stock solution was added to 49 ml of Ringer's solution to obtain 1/5000 working solution, which was placed in a brown bottle for use. It is best to use it now to maintain sufficient oxidizing power.

31/3000 neutral red solution: Weigh 0.5g neutral red dissolved in 50ml Ringer solution, dissolve it slightly (30-40°C), filter with filter paper, and store in brown bottle in dark place. Before use, 1 ml of 1% stock solution was added to 29 ml of Ringer's solution to obtain 1/3000 working solution, which was placed in a brown bottle for use.

3 Materials: human oral epithelial cells, onion bulbs, epidermal cells, mung bean root tips

experimental method

(1) Hyperactive staining and observation of mitochondria

Supervisible staining and observation of mitochondria in oral mucosal epithelial cells

The slide was heated at 37 ° C in a constant temperature water bath:

1/5000 Jenus Green B solution 2 drops

Human oral epithelial cell mucus toothpick scraping

Dyeing for 10-15 min, cover the coverslip, aspirate the surrounding dye solution, observe under the microscope

2 Super-staining and observation of mitochondria in epidermal cells of onion bulbs

The slide was heated at 37 ° C in a constant temperature water bath:

1/5000 Jenus Green B solution 1 drop

Peeling of epidermis in onion bulbs

Dyeing for 10-15 min, aspirate the dye solution, add 1 drop of Ringer solution, cover the coverslip, observe under the microscope

(2) Neutral red staining of the vacuolar system of the root tip of mung bean

The slide was heated at 37 ° C in a constant temperature water bath:

Sliced ​​longitudinal section of nascent mung bean seedlings (1-2 cm)

1/3000 neutral red solution 1 drop

Dyeing for 5-10 min, aspirate the dye solution, add 1 drop of Ringer solution, cover with a cover slip and flatten the root tip, observe under the microscope

今日推荐阅读 线粒体和液泡系的超活染色与观察

Microscope

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